Aberrant interleukin family cytokine activation of inflammation characterizes a wide spectrum of autoimmune, inflammatory, neoplastic, and neonatal disorders, including arthritis, obstructive lung disease, psoriasis and asthma. We created a novel technology, protein painting, and used it to discover, and create, a novel peptide inhibitor, called Arg286p, that abolishes the necessary interaction of IL1-RAcP with the IL-1?-IL1Receptor complex. Arg286p constitutes a novel specific and potent approach to treat interleukin mediated inflammatory diseases. The binding interface contact points between interacting protein partners are important drug targets for the next generation of therapies that block such interactions. Employing a new class of small molecule affinity chemistry, protein painting has a positive hit specificity of 93% and yield up to ten fold higher compared to conventional cross linking or deuterium exchange methods. Using our technology to study the 3-way interaction of interleukin-1? (IL-1?, IL-1 receptor I (IL-1RI), and IL-1 receptor accessory protein (IL-1RAcP), we identified a highly conserved beta-loop Arg286 region of the IL-1RAcP protein that participates in a multivalent interaction with the ligand and the receptor. We validated this novel target to be necessary for IL1?-mediated signaling. We created a folded protein peptide Arg286p that corresponds to the Arg286 beta loop domain, and a monoclonal antibody that recognizes the IL-1RAcP Arg286 surface domain. Our Arg286 peptide compound abolished interleukin signaling in a cell culture model in a dose-dependent manner, compared to a control peptide lacking the arginine in the 286 position. The Arg286 peptide and the anti-Arg286 mAb also abolished formation of the IL-1?, IL-1RI, and IL-1RAcP 3-way complex, in vitro, in a dose-dependent manner. IL1-RAcP is an accessory protein that is recruited after the interleukin ligand binds to the receptor, and signaling requires all members of this three-way complex formation. Our new inhibitor Arg286p mimics a small key multivalent interaction region at the arginine 286 beta loop of IL1-RacP, thereby abolishing complex formation and preventing downstream inflammatory signaling. Since Arg286p acts downstream of receptor-ligand binding, it can massively amplify the potency for interleukin therapy even in the face of excess ligand, and may be superior to, or synergistic with, existing IL-1 therapies that competitively target the ligand. Under the Aims, we will create new structural modifications in this lead compound to optimize its affinity and stability for IL-1 and we will use it as a basis to create novel inhibitos specific for IL33 and IL36, for which clinical inhibitors do not exist. We will employ our protein painting technology in a novel iterative workflow to guide the design of peptide modulators (agonists and antagonists) that are either optimized in potency for IL-1 or specific for IL-33 and IL-36 in vitro, in three types of assays. The optimized version of our lead compound will be studied in a well-established animal model of osteoarthritis. Osteoarthritis causes suffering for 27 million Americans.